The present invention relates to the production of porcine circovirus type 2 (PCV2). More particularly, the invention relates to continuous cell lines that are highly susceptible to infection with PCV2 and to methods for the production of PCV2 using the cell lines.
Porcine circovirus (PCV) is a small, non-enveloped, circular, single-stranded DNA virus classified in the Circoviridae family. Murphy, F A., Fauquet, C M., Bishop, D H L., Ghabrial, S A., Jarvis, A W., Martelli, G P.; Mayo, M A., Summers, M D. Virus taxonomy. Sixth report of the International Committee on Taxonomy of Viruses. New York, N.Y: Springer-Verlag; 1995. pp. 166-168. It was originally identified and described as a contaminant of a porcine kidney cell line. Tischer, I., Gelderblom, H., Vettermann, W., Koch, M. A. A very small porcine virus with circular single-stranded DNA. Nature. 1982:295:64-66. Recently, PCV has been associated with a disease of pigs, the post-weaning multi-systemic wasting syndrome (PMWS), first observed in Western Canada. Ellis, J., Hassard, L., Clark, E., Harding, J., Allan, G., Willson, P., Strokappe, J., Martin, K., McNeilly, F., Meehan, F., Todd, D., Haines, D. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can. Vet. J., 1998; 39:44-51; Harding, J. C. S., Clark, E. G. Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS). Swine Health Prod. 1997; 5:201-203; Jue Liu, Isabelle Chen, and Jimmy Kwang, J. Virol. 2005: 79(13); 8262-74. PWMS has emerged as a major disease that poses a significant threat to the economics of the global swine industry. After its first appearance in Canada, PMWS has now spread to the United States, Europe and Asia. The syndrome mainly affects pigs between 6 and 14 weeks of age. It tends to be slow and progressive with a high fatality rate in affected pigs. See http:www dot aphis dot usda dot gov/vs/ceah backslash dei/taf backslash emergingdiseasenotice_files/pmws—0301.htm.
The clinical signs of PMWS are quite variable. Affected pigs may show signs of chronic wasting, respiratory distress, diarrhea, incoordination, paralysis, pale skin color and blue ears. Pigs usually demonstrate a decrease in growth rate and, occasionally, jaundice.
The diagnosis of PMWS is based on the age of affected pigs, typical wasting appearance and necropsy lesions. Microscopic and immunohistochemical examination of tissues reveals unique lung and lymphoid tissue lesions with the presence of PCV2. Id.
Antibacterial medication is usually ineffective in treating PWMS and currently no vaccines are available. Prevention of the syndrome is based on biosecurity precautions and good husbandry practices.
PCV2 has also been found in association with other diseases including porcine dermatitis and nephropathy syndrome (“PDNS”), congenital tremors (CT-All) reproductive disorders, prenatal myocarditis and proliferative and necrotizing pneumonia.
Vaccines employing PCV2 antigens have shown some initial success in preventing the PMWS. Fenaux, M., et al., A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs. J. Virol., 2004. 78(12): p. 6297-303; Blanchard, P. et al., Protection contre la maladie d'amaigrissement du porcelet (MAP) par vaccins a ADNet proteines recombinantes. Journees de la Recherche Porcine en France, 2004. 36: p. 345-352; Blanchard, P., et al., Protection of swine against porcine multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins. Vaccine, 2003. 21: p. 4565-4575; Pogranichniy, R. et al. Efficacy of inactivated PCV2 vaccines for preventing PMWS in CDCD pigs. American Association of Swine Veterinarians. 2004. Des Moines, Iowa. However, an effective vaccine is not currently available.
The development of vaccines, diagnostic agents and therapies for PMWS and other diseases associated with PCV2 viral infections will require efficient and reliable means for producing the virus in substantial quantities. PCV2 virus stocks have conventionally been produced by culturing the virus in porcine kidney cell-line PK15. The virus titers yielded from PK15 cell cultures, expressed as 50% tissue culture infectious dosage (“TCID50”) per milliliter, usually ranged from 104-105 and could never exceed 105. Immunofluorescence stainings of infected PK15 cell cultures have revealed that only about 40% of the cell population is susceptible to the PCV2 infection.
A need exists for a continuous cell line that is highly permissive to PCV2 infection and that reliably produces virus in high titers over extended periods of time.